The present invention relates to a device for analyzing substances by means of thin layer chromatography.
During the analysis of substances by means of thin layer chromatography, the substance to be analyzed is dropped or applied onto a thin layer plate, which has a support plate and, arranged thereon, a thin layer of a functional substance, for example silica gel, C18, C8, C4, C2, C6H5, NH2, Al2O3, paper (cellulose), starch, etc. The thin layer plate is then placed in a suitable solvent, or generally a mixture of solvents, for example hexane, hexane and ethyl acetate in the ratio 1:2, cyclohexane and dichloromethane in the ratio 2:1 etc., which, because of the capillary action of the thin layer, migrates upward and entrains upward the substance to be analyzed that has been dropped on or applied, or at least various components of it. Since the various substances or components migrate upward over different distances, information can be obtained therefrom about the substance or the components, especially if a reference substance has likewise been applied.
To date, the analysis of substances by means of thin layer chromatography has usually been carried out manually. In this case, the thin layer plate is placed flat on the laboratory table and a drop of a reference substance and drop of the substance to be analyzed or, respectively next to one another, a drop of various substances to be analyzed, are applied onto the thin layer plate on a start line, for example using a pipette. The thin layer plate is then placed manually in a trough containing solvent, so that the lower part of the thin layer plate is wetted and the solvent can migrate upward, and in doing so it entrains upward the substance to be analyzed, or at least various components of it. So that the solvent on the thin layer plate does not evaporate too quickly, the solvent trough and the thin layer plate are covered with a trough cover. Before the solvent reaches the top of the thin layer plate, the latter is withdrawn from the solvent trough.
The analysis of substances by means of manual thin layer chromatography has the disadvantage that it is relatively elaborate and requires the presence of a chemist or laboratory assistant, which can be problematic especially since the upward migration of the solvent may last several hours, and the thin layer plate may possibly need to be withdrawn from the solvent trough in the middle of the night so that the result image does not become smeared.
These disadvantages can be partially avoided by means of the Automatic Developing Chamber xe2x80x9cADCxe2x80x9d from the company Camag, CH-4132 Muttenz, which comprises a solvent trough with a solvent channel, a trough cover and means for automatically supplying and withdrawing solvent. The thin layer plate provided with a substance to be analyzed is placed in the developing chamber, the developing chamber is closed and, finally, solvent is automatically supplied and subsequently withdrawn. The substance to be analyzed, however, still needs to be dropped or applied onto the thin layer plate outside the automatic developing chamber, and the thin layer plate then needs to be picked up and placed in the developing chamber.
In view of the disadvantages of the previously known methods and devices described above for the analysis of substances by means of thin layer chromatography, the object of the invention is as follows. A device is to be provided for the analysis of substances by means of thin layer chromatography of the type mentioned in the introduction, which permits simplified, automatable conduct of the substance analysis.
The essence of the invention is that a device for analyzing substances by means of thin layer chromatography, with a solvent trough which has at least one solvent channel, has means which make it possible to arrange a thin layer plate standing obliquely with respect to the horizontal in the solvent channel so that a substance to be analyzed can be dropped or applied onto the thin layer plate from above, and solvent can be supplied to the solvent channel at a solvent supply point so that the upward migration of components of the substance to be analyzed can be started without the position of the thin layer plate needing to be changed for this purpose.
Owing to the fact that the thin layer plate is arranged obliquely with respect to the horizontal, its position does not need to be changed throughout the whole time, which simplifies the entire procedure and allows it to be automated without great outlay. Automation is furthermore facilitated by the fact that the supply of a substance to be analyzed can take place from above, and commercially available robots that can be moved in one or two horizontal directions and the vertical direction can hence be used for this. Since these robots generally also carry out steps for the preceding and subsequent processes, it is highly advantageous to use them as well for the analysis of substances by means of thin layer chromatography, which may also be done on-line.
In a preferred embodiment, the solvent can furthermore be supplied from above.
The optimum angle xcex1 between the thin layer plate and the horizontal is between 40xc2x0 and 50xc2x0, preferably 45xc2x0. At such an angle, on the one hand, the substance to be analyzed can easily be dropped on or applied from above and, on the other hand, the lower part of the thin layer plate can be wetted in a suitable way with the solvent, which can migrate upward properly together with the substance to be analyzed or components of it. In general, an angle xcex1 between 20xc2x0 and 80xc2x0, or even between 5xc2x0 and 85xc2x0, is suitable, while an angle xcex1 less than or equal to 5xc2x0 and greater than 1xc2x0, or greater than or equal to 85xc2x0 and less than 89xc2x0, is albeit less suitable but nevertheless possible.
Advantageously, the solvent trough has two or more mutually independent solvent channels, with at least two thin layer plates preferably being arrangeable in a solvent channel. It is also entirely feasible to implement ten or more solvent channels, with each feeding solvent to one thin layer plate. A plurality of substances can then be analyzed using two or more different solvents.
In a preferred embodiment, the or each solvent channel is inclined toward a solvent extraction point, which preferably coincides with the solvent supply point. This makes it possible not only to supply solvent at a single point, but also to subsequently extract solvent at a single point, preferably the same point. The migration process is started when the solvent is supplied, and it is terminated when the solvent is withdrawn.
In the normal case, the device according to the invention furthermore has a trough cover, which substantially seals the solvent channel or the solvent channels and the thin layer plate or plates from the outside. In this way, it is possible to produce a saturated atmosphere, which prevents excessively rapid evaporation of the solvent or solvents, especially if the vapor pressure is high at the analysis temperature.
Advantageously, the trough cover has at the top holes or slots or an open face, which make it possible to supply solvent and drop or apply substances to be analyzed onto the thin layer plate or plates through the trough cover, with the holes, slots or the open face preferably being closed by septa or a septum plate. The substance to be analyzed and solvents can hence be supplied from above when the solvent trough is covered, with sealed coverage being ensured by the septa or septum plates that are present if applicable.
Normally, the device according to the invention also comprises a delivery instrument, which preferably comprises a hollow needle that can be moved vertically and in at least one horizontal direction, by which substances to be analyzed can be dropped or applied automatically onto the thin layer plate or plates and solvents can be supplied automatically at the solvent supply point or points and can be extracted automatically at the solvent extraction point or points. The delivery instrument permits automatic handling of the substances and solvents so that, after the control unit of the delivery instrument has been programmed, the entire method can proceed without a chemist or laboratory assistant needing to intervene.
In a preferred embodiment, the device according to the invention has a reagent matrix, which comprises reception holes for reagent containers and delivery holes passing through between them, through which a delivery and withdrawal tool for delivering and withdrawing substances to be analyzed and solvents can be fed. Reagent containers containing substances to be analyzed can then simply be arranged in the reception holes and, for the analysis, a substance to be analyzed can be withdrawn from the reagent container and applied onto a thin layer plate arranged underneath through a delivery hole. In the normal case, solvents are withdrawn from separate large containers, although they may also be supplied in a different way instead of with said delivery and withdrawal tool, e.g. by pumping, opening a valve etc.
Advantageously, there are means for visualizing the components of the substance to be analyzed that have migrated upward, which preferably comprise a UV lamp and/or means for spraying with chemical reagents. The analysis result can hence be made directly visible.
In a preferred embodiment, the device according to the invention is furthermore provided with means for determining the time at which the solvent that is present needs to be withdrawn from the solvent channel or channels, which preferably comprise a CCD camera. The optimum time for withdrawing the solvent can hence be determined automatically, and the solvent can then be withdrawn automatically, without a chemist or laboratory assistant needing to be present for this purpose. Furthermore, in this embodiment, the time for withdrawing the solvent does not need to be pre-programmed, i.e. estimated.
The device according to the invention for the analysis of substances by means of thin layer chromatography will be described in more detail below with reference to the appended drawings with the aid of seven exemplary embodiments.